1. Field of the Invention
The present invention relates to a novel growth factor for vascular endothelial cells identified in media condition of cultured bovine pituitary follicular cells and of murine tumor cells. The invention also relates to isolation and purification of the growth factor.
2. Description of the Problem and Related Art
Numerical references in parenthesis in the text refer to the publications listed below in the Reference Section.
Angiogenesis is a multi-step phenomenon which involves capillary endothelial cell proliferation, migration and tissue infiltration (1). It plays a central role in a variety of physiological and pathological processes such as embryonic development, wound healing, atherosclerosis and tumor growth (1,2). Several factors induce angiogenesis have recently been isolated and characterized. Among these are the acidic and basic form of fibroblast growth factor (FGF), both capable of stimulating capillary endothelial cell growth in vitro as well as being chemotactic for that cell type (2). In addition, both acidic and basic FGF stimulate collagenase activity and plasminogen activator production while blocking the activity of plasminogen inhibitor (3,4). These enzymes are involved in the breakdown of the capillary basement membrane, an event required in order for angiogenesis to take place (1). Other growth factors such as tumor necrosis factor alpha (TNFα), transforming growth factor beta (TGF), transforming growth factor alpha (TGFα), and epidermal growth factor (EGF) are also angiogenic in vivo (5-8). However, with the exception of TGFα and EGF at high concentrations (7), these growth factors are not mitogenic for capillary endothelial cells (5,6); their action on the angiogenic process is therefore probably indirect, resulting from such activities as the attraction of macrophages by chemotaxis (9,10) which in turn release direct angiogenic factor(s), one of which could be basic FGF (11).
A number of growth factors, such as acidic and basic FGF, PDGF and EGF, are broadly mitogenic for a number of cell types. This broad mitogenicity is desirable in many types of wound healing applications. There are, however, specific types of wound healing applications in which it would be more desirable to employ growth factors having more cell-specific mitogenic activity. For example, following vascular graft surgery or balloon angioplasty, it would be highly desirable to employ a wound healing agent incorporating mitogenic factor having mitogenic activity that is highly specific for vascular endothelial cells. At present, no highly suitable mitogenic factor exists for this type of application.
In the course of our studies on the localization of basic FGF in various tissues, it was observed that, in the pituitary gland, folliculo stellate cells are the main producers of bFGF (18). Although the medium conditioned by those cells was found to be strongly mitogenic for capillary endothelial cells, little if any bFGF is present in it, thus suggesting that, in addition to synthesizing bFGF, these cells are also capable of producing another endothelial cell mitogen. To date, however, this mitogenic activity has not been purified or characterized.
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All of the references and/or patents cited in this application are incorporated herein by reference.
Previously published research describes the culture of homogeneous populations of bovine pituitary follicular or folliculo-stellate cells (FC) (20) and subsequently characterized them as ion transport elements, possibly involved in the regulation of ion composition and osmolarity of the interstitial fluid in the adenohypophysial cell cords (41, 42). It is also reported that FC produce the angiogenic mitogen basic fibroblast growth factor (bFGF) (17).
The gene for bFGF (43), similarly to the gene for acidic fibroblast growth factor (aFGF) (13), does not code for a conventional signal peptide, required for the extracellular transport of proteins according to classic secretory pathways (44). Accordingly, the growth factor is not appreciably secreted in the medium (15,45) and responsive cell types are dependent on exogenous bFGF for optimal proliferation in culture, even though they may contain significant intracellular concentrations of mitogen (46,47,48).
It was initially observed, however, that the medium conditioned by bovine pituitary FC is mitogenic for adrenal-cortex-derived capillary endothelial cells. Interestingly, these cells are responsive either to bFGF or aFGF but are not stimulated to proliferate by EGF, TGF alfa, TGF beta, PDGF, insulin or TNF (2). These observations led us to consider the possibility that an endothelial cell growth factor distinct from FGF and possibly any other known growth factor may be secreted by cultured FC.
The present invention describes the purification and biological characterizations of such a novel growth factor. Its unique N-terminal amino acid sequence, as well as its specificity for vascular endothelial cells, distinguishes it from any previously described growth factor.